Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet.

UNLABELLED: The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. SIGNIFICANCE STATEMENT: Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility.

Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function.

The adipocyte-derived hormone leptin acts in the brain to modulate the central driver of fertility: the gonadotropin releasing hormone (GnRH) neuronal system. This effect is indirect, as GnRH neurons do not express leptin receptors (LEPRs). Here we test whether GABAergic or glutamatergic neurons provide the intermediate pathway between the site of leptin action and the GnRH neurons. Leptin receptors were deleted from GABA and glutamate neurons using Cre-Lox transgenics, and the downstream effects on puberty onset and reproduction were examined. Both mouse lines displayed the expected increase in body weight and region-specific loss of leptin signaling in the hypothalamus. The GABA neuron-specific LEPR knock-out females and males showed significantly delayed puberty onset. Adult fertility observations revealed that these knock-out animals have decreased fecundity. In contrast, glutamate neuron-specific LEPR knock-out mice displayed normal fertility. Assessment of the estrogenic hypothalamic-pituitary-gonadal axis regulation in females showed that leptin action on GABA neurons is not necessary for estradiol-mediated suppression of tonic luteinizing hormone secretion (an indirect measure of GnRH neuron activity) but is required for regulation of a full preovulatory-like luteinizing hormone surge. In conclusion, leptin signaling in GABAergic (but not glutamatergic neurons) plays a critical role in the timing of puberty onset and is involved in fertility regulation throughout adulthood in both sexes. These results form an important step in explaining the role of central leptin signaling in the reproductive system. Limiting the leptin-to-GnRH mediators to GABAergic cells will enable future research to focus on a few specific types of neurons.

RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action.

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

Leptin deficiency and diet-induced obesity reduce hypothalamic kisspeptin expression in mice.

The hormone leptin modulates a diverse range of biological functions, including energy homeostasis and reproduction. Leptin promotes GnRH function via an indirect action on forebrain neurons. We tested whether leptin deficiency or leptin resistance due to a high-fat diet (HFD) can regulate the potent reproductive neuropeptide kisspeptin. In mice with normalized levels of estradiol, leptin deficiency markedly reduced kisspeptin gene expression, particularly in the arcuate nucleus (ARC), and kisspeptin immunoreactive cell numbers in the rostral periventricular region of the third ventricle (RP3V). The HFD model was used to determine the effects of diet-induced obesity and central leptin resistance on kisspeptin cell number and gene expression. DBA/2J mice, which are prone to HFD-induced infertility, showed a marked decrease in kisspeptin expression in both the RP3V and ARC and cell numbers in the RP3V after HFD. This is the first evidence that kisspeptin can be regulated by HFD and/or increased body weight. Next we demonstrated that leptin does not signal (via signal transducer and activator of transcription 3 or 5, or mammalian target of rapamycin) directly on kisspeptin-expressing neurons in the RP3V. Lastly, in leptin receptor-deficient mice, neither GnRH nor kisspeptin neurons were activated during a preovulatory-like GnRH/LH surge induction regime, indicating that leptin's actions on GnRH may be upstream of kisspeptin neurons. These data provide evidence that leptin's effects on reproductive function are regulated by kisspeptin neurons in both the ARC and RP3V, although in the latter site the effects are likely to be indirect.

Leptin indirectly regulates gonadotropin-releasing hormone neuronal function.

The adipose-derived hormone leptin communicates information about metabolic status to the hypothalamic GnRH neuronal system. It is unclear whether leptin can act directly on GnRH neurons. To examine this, we used three approaches. First, the presence of leptin-induced signal transducer and activator of transcription-3 activation was examined in GnRH neurons in male and female rats. Intracerebroventricular treatment with 4 mug leptin-induced robust signal transducer and activator of transcription-3 expression within the anteroventral periventricular nucleus but not in GnRH neurons. Second, fertility was assessed in male and female CRE-loxP transgenic mice with conditional leptin receptor (Lepr) deletion from either all forebrain neurons or GnRH neurons only. Forebrain neuron LEPR deletion prevented the onset of puberty resulting in infertility in males and females and blocked estradiol-induced LH surge. However, mice with GnRH neuron-selective Lepr deletion exhibited normal fertility apart from a slight puberty delay in males. Lastly, the highly sensitive technique of single-cell nested PCR was used to test for Lepr transcript presence in individual GnRH neurons, identified in situ using GnRH-green fluorescent protein transgenics. Whereas 75% of positive control (proopiomelanocortin) neurons contained Lepr mRNA, no (none of 18) GnRH neurons were Lepr mRNA positive. Collectively, these results show that leptin does not act directly on GnRH neurons in rats and mice. Leptin appears to regulate GnRH function via forebrain neurons that are afferent to GnRH because forebrain neuronal LEPR deletion caused infertility. The location and phenotype of these leptin-responsive neurons remains to be elucidated.